Nanoparticle Classification in Wide-field Interferometric Microscopy by Supervised Learning from Model

Interference enhanced wide-field nanoparticle imaging is a highly sensitive technique that has found numerous applications in labeled and label-free sub-diffraction-limited pathogen detection. It also provides unique opportunities for nanoparticle classification upon detection. More specif- ically, the nanoparticle defocus images result in a particle-specific response that can be of great utility for nanoparticle classification, particularly based on type and size. In this work, we com- bine a model based supervised learning algorithm with a wide-field common-path interferometric microscopy method to achieve accurate nanoparticle classification. We verify our classification schemes experimentally by using gold and polystyrene nanospheres.Comment: 5 pages, 2 figure

Interferometric reflectance microscopy for physical and chemical characterization of biological nanoparticles

Biological nanoparticles have enormous utility as well as potential adverse impacts in biotechnology, human health, and medicine. The physical and chemical properties of these nanoparticles have strong implications on their distribution, circulation, and clearance in vivo. Accurate morphological visualization and chemical characterization of nanoparticles by label-free (direct) optical microscopy would provide valuable insights into their natural and intrinsic properties. However, three major challenges related to label-free nanoparticle imaging must be overcome: (i) weak contrast due to exceptionally small size and low-refractive-index difference with the surrounding medium, (ii) inadequate spatial resolution to discern nanoscale features, and (iii) lack of chemical specificity. Advances in common-path interferometric microscopy have successfully overcome the weak contrast limitation and enabled direct detection of low-index biological nanoparticles down to single proteins. However, interferometric light microscopy does not overcome the diffraction limit, and studying the nanoparticle morphology at sub-wavelength spatial resolution remains a significant challenge. Moreover, chemical signature and composition are inaccessible in these interferometric optical measurements. This dissertation explores innovations in common-path interferometric microscopy to provide enhanced spatial resolution and chemical specificity in high-throughput imaging of individual nanoparticles. The dissertation research effort focuses on a particular modality of interferometric imaging, termed “single-particle interferometric reflectance (SPIR) microscopy”, that uses an oxide-coated silicon substrate for enhanced coherent detection of the weakly scattered light. We seek to advance three specific aspects of SPIR microscopy: sensitivity, spatial resolution, and chemical specificity. The first one is to enhance particle visibility via novel optical and computational methods that push optical detection sensitivity. The second one is to improve the lateral resolution beyond the system's classical limit by a new computational imaging method with an engineered illumination function that accesses high-resolution spatial information at the nanoscale. The last one is to extract a distinctive chemical signature by probing the mid-infrared absorption-induced photothermal effect. To realize these goals, we introduce new theoretical models and experimental concepts. This dissertation makes the following four major contributions in the wide-field common-path interferometric microscopy field: (1) formulating vectorial-optics based linear forward model that describes interferometric light scattering near planar interfaces in the quasi-static limit, (2) developing computationally efficient image reconstruction methods from defocus images to detect a single 25 nm dielectric nanoparticle, (3) developing asymmetric illumination based computational microscopy methods to achieve direct morphological visualization of nanoparticles at 150 nm, and (4) developing bond-selective interferometric microscopy to enable multispectral chemical imaging of sub-wavelength nanoparticles in the vibrational fingerprint region. Collectively, through these research projects, we demonstrate significant advancement in the wide-field common-path interferometric microscopy field to achieve high-resolution and accurate visualization and chemical characterization of a broad size range of individual biological nanoparticles with high sensitivity

High-resolution Imaging of nanoparticles in wide-field interferometric scattering microscopy

Single particle interferometric scattering microscopy has demonstrated great capability in label-free imaging of sub-wavelength dielectric nanoparticles (r<25 nm); however, it suffers from diffraction-limited resolution. Here, we demonstrate ~2-fold improvement in lateral resolution upon asymmetric illumination.Published versio

Interferometric detection and enumeration of viral particles using Si-based microfluidics

Single-particle interferometric reflectance imaging sensor enables optical visualization and characterization of individual nanoparticles without any labels. Using this technique, we have shown end-point and real-time detection of viral particles using laminate-based active and passive cartridge configurations. Here, we present a new concept for low-cost microfluidic integration of the sensor chips into compact cartridges through utilization of readily available silicon fabrication technologies. This new cartridge configuration will allow simultaneous detection of individual virus binding events on a 9-spot microarray, and provide the needed simplicity and robustness for routine real-time operation for discrete detection of viral particles in a multiplex format.This work was supported in part by a research contract with the ASELSAN Research Center, Ankara, Turkey, and in part by the European Union's Horizon 2020 FET Open program under Grant 766466-INDEX. (ASELSAN Research Center, Ankara, Turkey; 766466-INDEX - European Union's Horizon 2020 FET Open program)First author draf

Computational nanosensing from defocus in single particle interferometric reflectance microscopy

Single particle interferometric reflectance (SPIR) microscopy has been studied as a powerful imaging platform for label-free and highly sensitive biological nanoparticle detection and characterization. SPIR's interferometric nature yields a unique 3D defocus intensity profile of the nanoparticles over a large field of view. Here, we utilize this defocus information to recover high signal-to-noise ratio nanoparticle images with a computationally and memory efficient reconstruction framework. Our direct inversion approach recovers this image from a 3D defocus intensity stack using the vectorial-optics-based forward model developed for sub-diffraction-limited dielectric nanoparticles captured on a layered substrate. We demonstrate proof-of-concept experiments on silica beads with a 50 nm nominal diameter.Accepted manuscript2021-12-0

Bond-selective interferometric scattering microscopy

Interferometric scattering microscopy has been a very promising technology for highly sensitive label-free imaging of a broad spectrum of biological nanoparticles from proteins to viruses in a high-throughput manner. Although it can reveal the specimen's size and shape information, the chemical composition is inaccessible in interferometric measurements. Infrared spectroscopic imaging provides chemical specificity based on inherent chemical bond vibrations of specimens but lacks the ability to image and resolve individual nanoparticles due to long infrared wavelengths. Here, we describe a bond-selective interferometric scattering microscope where the mid-infrared induced photothermal signal is detected by a visible beam in a wide-field common-path interferometry configuration. A thin film layered substrate is utilized to reduce the reflected light and provide a reference field for the interferometric detection of the weakly scattered field. A pulsed mid-IR laser is employed to modulate the interferometric signal. Subsequent demodulation via a virtual lock-in camera offers simultaneous chemical information about tens of micro- or nano-particles. The chemical contrast arises from a minute change in the particle's scattered field in consequence of the vibrational absorption at the target molecule. We characterize the system with sub-wavelength polymer beads and highlight biological applications by chemically imaging several microorganisms including Staphylococcus aureus, Escherichia coli, and Candida albicans. A theoretical framework is established to extend bond-selective interferometric scattering microscopy to a broad range of biological micro- and nano-particles.First author draf

Background-suppressed high-throughput mid-infrared photothermal microscopy via pupil engineering

First author draf

Patient and physician delay in the diagnosis and treatment of non-small cell lung cancer in Turkey

Aim: The early diagnosis and treatment of lung cancer are important for the prognosis of patients withlung cancer. This study was undertaken to investigate patient and doctor delays in the diagnosis andtreatment of NSCLC and the factors affecting these delays.Materials and methods: A total of 1016 patients, including 926 (91.1%) males and 90 (8.9%) females with amean age of 61.5 10.1 years, were enrolled prospectively in this study between May 2010 and May2011 from 17 sites in various Turkish provinces.Results: The patient delay was found to be 49.9 96.9 days, doctor delay was found to be 87.7 99.6 days,and total delay was found to be 131.3 135.2 days. The referral delay was found to be 61.6 127.2 days,diagnostic delay was found to be 20.4 44.5 days, and treatment delay was found to be 24.4 54.9 days.When the major factors responsible for these delays were examined, patient delay was found to be morefrequent in workers, while referral delay was found to be more frequent in patients living in villages(p < 0.05). We determined that referral delay, doctor delay, and total delay increased as the number of doctorswho were consulted by patients increased (p < 0.05). Additionally, we determined that diagnostic andtreatment delays were more frequent at the early tumour stages in NSCLC patients (p < 0.05)

High-throughput, high-resolution interferometric light microscopy of biological nanoparticles

Label-free, visible light microscopy is an indispensable tool for studying biological nanoparticles (BNPs). However, conventional imaging techniques have two major challenges: (i) weak contrast due to low-refractive-index difference with the surrounding medium and exceptionally small size and (ii) limited spatial resolution. Advances in interferometric microscopy have overcome the weak contrast limitation and enabled direct detection of BNPs, yet lateral resolution remains as a challenge in studying BNP morphology. Here, we introduce a wide-field interferometric microscopy technique augmented by computational imaging to demonstrate a 2-fold lateral resolution improvement over a large field-of-view (>100 × 100 μm2), enabling simultaneous imaging of more than 104 BNPs at a resolution of ∼150 nm without any labels or sample preparation. We present a rigorous vectorial-optics-based forward model establishing the relationship between the intensity images captured under partially coherent asymmetric illumination and the complex permittivity distribution of nanoparticles. We demonstrate high-throughput morphological visualization of a diverse population of Ebola virus-like particles and a structurally distinct Ebola vaccine candidate. Our approach offers a low-cost and robust label-free imaging platform for high-throughput and high-resolution characterization of a broad size range of BNPs.Accepted manuscrip